The present invention relates to therapeutic and diagnostic methods and compositions based on Jagged/Notch proteins and nucleic acids, and on the role of their signaling pathway in endothelial cell migration, angiogenesis, and/or differentiation.
The functional integrity of the human vascular system is maintained by the endothelial cell which monitors the non-thrombogenic interface between blood and tissue in vivo. Thus, factors that influence human endothelial cell function may contribute significantly to the regulation and maintenance of homeostasis (see Maciag, 1984, In: Progress in Hemostasis and Thrombosis, pp. 167-182, Spaet, ed., A. R. Liss, New York; Folkman and Klagsburn, 1987, Science 235:442-447; Burgess and Maciag, 1989, Annu. Rev. Biochem. 58:575-606). Likewise, events that perturb this complex equilibrium are relevant to the pathophysiology of human disease states in which cellular components of the vascular tree are active participants including, e.g., atherogenesis, coronary insufficiency, hypertension, rheumatoid arthritis, solid tumor growth and metastasis, and wound repair.
Since the endothelium is present in all organs and tissues, endothelial cell function is also fundamental to the physiology and integration of these multicellular systems. This includes the ability to monitor and interface with repair systems that employ the tightly regulated inflammatory, angiogenic and neurotropic responses. Indeed, biochemical signals that are responsible for the modification of these responses have been well characterized as polypeptide growth factors and cytokines; however, their mechanisms of operation have, prior to the present invention, been poorly understood, impeding their acceptance as valuable tools in clinical management.
A major accomplishment of modern biology has been the recognition that structural elements responsible for physiologic functions are conserved throughout the animal kingdom. Genetic analysis of yeast, C. elegans, Xenopus, Zebra fish, and Drosophila, among others, has provided new insight into the regulation of the cell cycle, organelle biosynthesis and trafficking, cell fate and lineage decisions during development, as well as providing the fundamental principles for transcriptional/translational/post-translational regulation. Indeed, the conservation of structure-function-principles exhibited by such systems has generated new insight into these and other regulatory systems utilized by mammalian cells. Moreover, a resolution of the genetic structure of the mammalian homologs for such genes in non-mammalian species has often led to a discernment of their function in mammals, even though the delineation of the function of a particular homologous mammalian gene or gene fragment may well be serendipitous. In many cases, it is the result produced by expression and differential cDNA cloning strategies that manifest mammalian DNA sequences with homology to genes previously identified in more primitive species.
During the past decade, differential cDNA cloning methods, including e.g., conventional subtractive hybridization (Hla and Maciag, 1990, Biochem. Biophys. Res. Commun. 167:637-643), differential polymerase chain reaction (PCR)-oriented hybridization (Hla and Maciag, 1990, J. Biol. Chem. 265:9308-9313), and more recently, a modification of the differential display (Zimrin et al., 1995, Biochem. Biophys. Res. Commun. 213:630-638) were used to identify genes induced during the process of human umbilical vein endothelial cell (HUVEC) differentiation in vitro. Very early studies disclosed that HUVEC populations are able to generate capillary-like, lumen-containing structures when introduced into a growth-limited environment in vitro (Maciag et al., 1982, J. Cell Biol. 94:511-520). These studies permitted the identification and characterization of protein components of the extracellular matrix as inducers of this differentiation process, while at the same time defining the capillary-like structures as non-terminally differentiated (Maciag, 1984, In: Progress in Hemostasis and Thrombosis, pp. 167-182, Spaet, ed., A. R. Liss, New York). Additional experiments have elucidated the importance of polypeptide cytokines, such as IL-1 (Maier et al., 1990, J. Biol. Chem. 265:10805-10808) and IFNγ (Friesel et al., 1987, J. Cell Biol. 104:689-696), as inducers of HUVEC differentiation in vitro, and ultimately lead to an understanding that the precursor form of IL-1α was responsible for the induction of HUVEC senescence in vitro (Maciag et al., 1981, J. Cell Biol. 91:420-426; Maier et al., 1990, Science 249:1570-1574)—the only truly terminal HUVEC phenotype identified to date as summarized in FIG. 1.
Recent research has employed differential cDNA cloning methods, which permits the identification of new and very interesting genes. However, until very recently, establishing their identity did not provide insight into the mechanism of HUVEC differentiation. Current research has focused upon the fibroblast growth factor (FGF) and interleukin (IL)-1 gene families as regulators of the angiogenesis process, both in vitro and in vivo (Friesel et al., 1995, FASEB J. 9:919-925; Zimrin et al., 1996, J. Clin. Invest. 97:1359). The human umbilical vein endothelial cell (HUVEC) has proven to be an effective model for studying the signal pathways utilized by FGF-1 to initiate HUVEC migration and growth, the role of IL-1α as an intracellular inhibitor of FGF-1 function and modifier of HUVEC senescence, and the interplay between the FGF and the IL-1 gene families as key effectors of HUVEC differentiation in vitro. Such insight has enabled the present inventors to use modem molecular methods to identify a key regulatory ligand-receptor signaling system, which is able to both induce capillary endothelial cell migration and repress large vessel endothelial cell migration.
The Jagged/Serrate/Delta-Notch/Lin/Glp signaling system, originally described during the development of C. elegans and Drosophila as an essential system instrumental in cell fate decisions, has been found to be highly conserved in mammalian cells (Nye and Kopan, 1995, Curr. Biol. 5:966-969). Notch proteins comprise a family of closely-related transmembrane receptors initially identified in embryologic studies in Drosophila (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). The genes encoding the Notch receptor show a high degree of structural conservation, and contain multiple EGF repeats in their extracellular domains (Coffman et al., 1990, Science 249:1438-1441; Ellisen et al., 1991, Cell 66:649-661; Weinmaster et al., 1991, Development 113:199-205; Weinmaster et al., 1992, Development 116:931-941; Franco del Amo et al., 1992, Development 115:737-744; Reaume et al., 1992, Dev. Biol. 154:377-387; Lardelli and Lendahi, 1993, Mech. Dev. 46:123-136; Bierkamp and Campos-Ortega, 1993, Mech. Dev. 43:87-100; Lardelli et al., 1994, Exp. Cell Res. 204:364-372). In addition to the thirty-six EGF repeats within the extracellular domain of Notch 1, there is a cys-rich domain composed of three Notch Lin Glp (NLG) repeats, which is important for ligand function, followed by a cys-poor region between the transmembrane and NLG domain.
The intracellular domain of Notch 1 contains six ankyrin/Cdc10 repeats positioned between two nuclear localization sequences (NLS) (Artavanis-Tsakonas et al., 1995, Science 268:225-232). This motif is found in many functionally diverse proteins (see, e.g., Bork, 1993, Proteins 17:363-374), including members of the Rel/NF-κB family (Blank et al., 1992, TIBS 17:135-140), and is thought to be responsible for protein-protein interactions. Notch has been shown to interact with a novel ubiquitously distributed cytoplasmic protein deltex through its ankyrin repeats, a domain shown by deletion analysis to be necessary for activity (Matsuno et al., 1995, Development 121:2633-2644).
Carboxy terminal to this region is a polyglutamine-rich domain (OPA) and a pro-glu-ser-thr (PEST) domain (SEQ ID NO:33) which may be involved in signaling protein degradation. There are numerous Notch homologs, including three Notch genes. (The corresponding structures for Lin-12 and Glp-1 are shown in FIG. 4.)
Several Notch ligands have been identified in vertebrates, including Delta, Serrate and Jagged. The Notch ligands are also transmembrane proteins, having highly conserved structures. These ligands are known to signal cell fate and pattern formation decisions through the binding to the Lin-12/Notch family of transmembrane receptors (Muskavitch and Hoffinann, 1990, Curr. Top. Dev. Biol. 24:289-328; Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; Greenwald and Rubin, 1992, Cell 68:271-281; Gurdon, 1992, Cell 68:185-199; Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247; and Weintraub, 1993, Cell 75:1241-1244). A related protein, the Suppressor of hairless (Su(H)), when co-expressed with Notch in Drosophila cells, is sequestered in the cytosol, but is translocated to the nucleus when Notch binds to its ligand Delta (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). Studies with constitutively activated Notch proteins missing their extracellular domains have shown that activated Notch suppresses neurogenic and mesodermal differentiation (Coffinan et al., 1993, Cell 73:659-671; Nye et al., 1994, Development 120:2421-2430).
The Notch signaling pathway (FIG. 3), which is apparently activated by Jagged in the endothelial cell, involves cleavage of the intracellular domain by a protease, followed by nuclear trafficking of the Notch fragment and the interaction of this fragment with the KBF2/RBP-Jk transcription factor (Jarriault et al., 1995, Nature 377:355-358; Kopan et al., 1996, Proc. Natl. Acad. Sci. USA 93:1683-1688), a homolog of the Drosophila Suppressor of hairless gene (Schweisguth et al., 1992, Cell 69:1199-1212), a basic helix-loop-helix transcription factor involved in Notch signaling in insects (Jennings et al., 1994, Development 120:3537-3548) and in the mouse (Sasai et al., 1992, Genes Dev. 6:2620-2634). This effector is able to repress the transcriptional activity of other genes encoding transcription factors responsible for entry into the terminal differentiation program (Nye et al., 1994; Kopan et al., 1994, J. Cell. Physiol. 125:1-9).
The Jagged gene encodes a transmembrane protein which is directed to the cell surface by the presence of a signal peptide sequence (Lindsell et al., 1995, Cell 80:909-917). While the intracellular domain contains a sequence with no known homology to intracellular regions of other transmembrane structures, the extracellular region of the ligand contains a cys-rich region, 16 epidermal growth factor (EGF) repeats, and a DSL (Delta Serrate Lag) domain. As shown in FIG. 2, the DSL domain as well as the EGF repeats, are found in other genes including the Drosophila ligands, Serrate (Baker et al., 1990, Science 250:1370-1377; Thomas et al., 1991, Development 111:749-761) and Delta (Kopczynski et al., 1988, Genes Dev. 2:1723-1735), and C. elegans genes Apx-1 (Henderson et al., 1994, Development 120:2913-2924; Mello et al., 1994, Cell 77:95-106) and Lag-2 (Tax et al., 1994, Nature 368:150-154).
Nevertheless, until the discovery of the presently disclosed invention, human Jagged remained undefined and the function and relationship, if any, of the human ligand to Notch remained unknown in the art. However, there was a recognized need in the art for a complete understanding of the protein's role in the regulation of cell differentiation and regulation. The present invention provides this understanding and in addition, provides compositions and methods useful for treatment of Jagged-related diseases in mammals.